Investigating the effect of a 3D physical microenvironment on hepatocyte structure, function, and adhesion signalling

Abstract

Presenting cells with a two -dimensional (2D) substrate, as is the case with traditional cell culture, causes them to aberrantly flatten out, and lose their characteristic cell shape. With the case of liver cells, their cuboidal cell shape is vital to cell -specific functions, such as xenobiotic metabolism. Accordingly, culturing hepatocytes in 2D may produce results that do not accurately reflect the behavior of such cells in-vivo. Cells in vivo are in constant contact with the ECM across three dimensions whereas culturing cells in 2D monolayers will alter the geometry of the cell leading to cytoskeletal remodeling and aberrant polarisation. As the cytoskeleton is physically and biochemically linked to the nucleus, this change in cell shape will in turn change the gene expression profile of the cell, leading to differences in cell behaviours such as proliferation, differentiation, and tissue -specific function. Mammalian cells respond to changes in the chemical composition and dimensionality of their microenvironment through complex signalling events at adhesion sites along their membrane. Changes in the microenvironment can result in up/down regulation of integrins, and changes in signalling downstream of adhesion.

Using a commercially available highly porous polystyrene scaffold, a method was developed to propagate cells continually in 3D. This model has been used to analyse how long -term growth under 3D conditions affects cytoskeletal organisation and whether adhesion signalling differs between 2D and 3D maintained cells. Cells maintained in 3D show significant cytoskeletal re - organisation and significant changes in cell morphology. 3D maintained cells generally adopt a more physiological morphology than 2D counterparts. These changes are amplified the longer the cells are maintained and propagated in 3D. In addition, these cells show a significant decrease in the phosphorylation of Focal Adhesion Kinase (FAK) and higher levels of α5β1.The differences in morphology and adhesion signaling between 2D and 3D maintained cells appear to lead to enhanced hepatic functionality. Under the conditions tested, 3D maintained HepG2s showed higher drug resistance to model xenobiotics, as well as generally higher levels of albumin, urea and glucose metabolism. 2D and 3D maintained cells also showed different levels of gene expression of key metabolic enzymes. As such, it could be argued that 3D propagation results in cells in vitro more closely reflecting the activity of their counterparts in vivo.